This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Palmitoylation is a lipid modification of proteins with the covalent attachment of a palmitoyl group to a cysteine residue through a thioester linkage. Palmitoylation is reversible and dynamic. Cycling between palmitoylation and depalmitoylation regulates important intracellular events. For example, membrane associated GTPase H-Ras shuttles among different subcellular compartments through regulation of palmitoylation turnover, inducing changes in its signaling transduction. Here we are developing a mass spectrometry method that directly detects palmitoylated proteins/peptides and identifies the palmitoylation sites. The model system chosen here is a synthetic palmitoylated peptide GDIFNQVVPRC(palm)PR. MALDI-TOF mass spectrum of the trypsin digest of the peptide showed partial loss of the palmitoyl group as evident by the presence of the CPR peptide. The fragmentation behavior of the palmitoylated peptide was investigated by tandem FTICR MS and tandem TOF experiments. Triply-charged peptide precursor ions were selected for low-energy CID and ECD analyses. Low-energy CID produced seven (out of the potential twelve) inter-residue cleavages. Contrary to a previous report (J. Mass Spectrom. 2006, 41, 229[unreadable]241), the palmitoyl group was retained in all CID fragments observed here. ECD produced a complete series of inter-residue cleavages except for that N-terminal to the proline residue. However, some ECD fragments were also present in their depalmitoylated form, albeit in low abundance. High-energy CID was also performed on the singly charged precursor ion on a MALDI-TOF/TOF instrument, which produced 10 inter-residue cleavages, and no palmitoyl group loss. Our results indicate that palmitoyl group losses could occur during trypsin digestion. Therefore, a top-down approach may be preferred for direct MS analysis of protein palmitoylation. Palmitoylation appeared to be stable under both CID and ECD, although ECD seemed to be the better method for palmitoylated site identification. We are currently applying this method to follow the palmitoylation in H-Ras produced by human aortic endothelial cells, using both top-down and bottom-up approaches, with emphasis on its role in apoptosis pathway under oxidative stress.